Identification of a Putative Sordarin Binding Site in Candida albicans Elongation Factor 2 by Photoaffinity Labeling

Abstract

Candida albicans EF-2 binds sordarin to a single class of binding sites with Kd = 1.26 μM. Equimolar mixtures of EF-2 and ribosomes, in the presence of a non-hydrolyzable GTP analog, reveal two classes of high affinity sordarin binding sites with Kd = 0.7 and 41.5 nM, probably due to the existence of two ribosome populations. Photoaffinity labeling of C. albicans EF-2 in the absence of ribosomes has been performed with [ 14C]GM258383, a photoactivatable sordarin derivative. Labeling is saturable and can be considered specific, because it can be prevented with another sordarin analog. The fragment Gln224-Lys232 has been identified as the modified peptide within the EF-2 sequence, Lys 228 being the residue to which the photoprobe was linked. This fragment is included within the G″-subdomain of EF-2. These results are discussed in the light of the high sordarin specificity toward fungal systems.